Stroke is currently a prevalent and high-risk condition among Veterans. Oligodendrocytes (OLs), abundant in white and grey matter, are the only myelin-forming cells in the brain. OL injury has profound consequences for the function of white matter, which is involved in almost all instances of ischemic stroke in humans. Also, OL damage could trigger secondary injury to neurons. Previously, more research was focused on neuroprotection, and many drugs for stroke treatment have been developed based on their ability to reduce neuronal damage in animal stroke models. However, clinical trial outcomes for neuroprotective drugs have been disappointing partly due to the drugs' failure to ameliorate ischemia/reperfusion (IR) damage to OLs. Therefore, we propose to investigate a novel mechanism of IR-induced OL injury for the design of effective therapeutics for cell protection and eventual stroke treatment. A hallmark of brain tissue injury in stroke is mitochondrial dysfunction and release of mitochondrial proteins, initiators of apoptosis. Although the mechanism behind the loss of mitochondrial integrity is obscure, ceramide, a membrane sphingolipid and an essential mediator of cell-stress responses, could be critical in brain IR-induced mitochondrial damage in OLs. Moreover, strong evidence implicates ceramide generation in response to cell- stress stimuli as a universal element of apoptosis. We have identified several isoforms of ceramide synthase, including CerS1, CerS2, and CerS6, localized in purified brain mitochondria. Our data suggest a novel mechanism of excessive ceramide accumulation in brain mitochondria due to selective activation of CerS6 after IR. We have also shown that synthetic analogs of natural ceramide inflict mitochondrial injury similar to that occurring in brain mitochondria after IR. This proposal tests the hypothesis that IR triggers activation of ceramide synthase CerS6 and ceramide accumulation in brain mitochondria leading to mitochondrial dysfunction and apoptotic OL death. The first specific aim is to determine the role of CerS6 in the IR-induced OL injury and brain damage. The second aim is to determine the mechanisms of CerS6/ceramide-mediated mitochondrial dysfunction in brain after IR. We will use an integrative approach by combining in vitro and in vivo studies in transgenic and knockout mice to accomplish the objectives. A powerful new methodology, tandem mass spectrometry, will be utilized for accurate detection of natural ceramide species and ceramide synthase activity. The studies designed in this proposal will establish the mitochondrial sphingolipid ceramide as a key molecule involved in OL injury and can provide the basis for development of groundbreaking therapeutics for stroke. It will improve the treatment and the quality of life of Veterans that will ultimately ameliorate the impact of the burden of care on the families and the American public.